Understanding the anatomic distribution of central cholinergic neurons will help to better understand how the brain normally functions, and how specific diseases affect brain function. Specific histochemical cholinergic markers are not presently available, although the enzyme choline acetyltransferase (ChAT) is selectively present in cholinergic neurons. The specificity of existing antisera raised against partially purified ChAT has been questioned. Therefore the proposed project will utilize "hybridoma" method to raise monoclonal antibodies against bovine ChAT. Spleen cells from immunized mice and rats will be fused with appropriate mouse myeloma cell lines. Clones of antiChAT-producing hybrid cells will be isolated by measuring the capacity of monoclonal antibodies to bind enzyme molecules in a double antibody co-precipitation assay. The monoclonal antibodies will be initially employed to map the immunohistochemical distribution of cholinergic neurons in adult rat brains and the ontogeny of these neurons in developing rat brains. Longer range experiments will be initiated to study cholinergic involvement in central nervous system diseases. Immunohistochemical studies of cholinergic neurons will be performed in Wobbler mice, an inbred strain which spontaneously develops clinicopathologic changes characteristic of motor neuron disease. Similar studies will be performed on post-mortem tissues obtained from human patients with motor neuron diseases, Huntington's Disease, and Alzheimer's Disease. The long range objectives of such studies are to better understand the pathogenesis of cholinergically-linked diseases in man.